PROJECT SUMMARY DNA replication is undeniably important for life and as a consequence, cells have evolved mechanisms to monitor replication fidelity and to coordinate completion of replication with other cell cycle events. The goal of this project is to understand how cells choreograph the duplication of their chromosomes, and how defects in DNA replication may contribute to some disorders in humans. Chromosome replication in eukaryotes is a process that involves the regulation of multiple initiation sites (origins) per chromosome that vary in their initiation timing (not all origins fire at the same time in S phase) and efficiency (not all origins fire in every cell cycle). Understanding how origin use is regulated is therefore critical for understanding how genome integrity is maintained. This notion is underscored by genetic disorders with links to replication defects: 1) Meier-Gorlin Syndrome with its point mutations in genes essential for origin licensing; 2) the mouse chaos3 mutation in a gene encoding part of the replicative helicase that is associated with breast cancer in mice; and 3) forms of replication fork errors that potentially contribute to human segmental copy number variants and autoimmune disorders such as lupus erythematosus. Yeast is an ideal model organism for studying DNA replication because of its small chromosomes, well defined origin sequences, ease of altering chromosome structure, and exceptional systems for genetic and genomic analysis. This project will address outstanding questions regarding why the choreography of chromosome replication is so important in eukaryotes: why do origins initiate replication at different times, what distinguishes origins in different temporal categories, what is the molecular basis for inefficient origins, and how do defects in replication origin firing and/or fork progression lead to genome instability and consequent disease states?